One objective of this research is to understand the biological significance of the breakdown and resynthesis of pyridine nucleotides in vivo (pyridine nucleotide cycles). There is evidence which suggests that such turnover cycles have different functions in prokaryotes and eukaryotes. In prokaryotes, we will investigate the possible contribution of DNA ligation to NAF turnover. A biochemical characterization of the enzyme NMN deamidase, which is involved in the NAD turnover pathway of Escherichia coli will be carried out. In eukaryotic cells, the biochemical pathway of NAD turnover in vivo will be determined, and the contribution of poly-ADPR synthesis and ADP-ribosylation of proteins to NAD turnover assessed. The fate of the ADPR moiety of NAD will be directly examined in vivo by microinjection techniques. These studies will be done using the human cell line D98/AH2 and/or Physarum polycephalum, a slime mold with a synchronous life stage. Finally, we plan to study regulation of NAD and NADP intracellular levels. Using Salmonella typhimurium, we will isolate mutants in all genetic loci involved in pyridine nucleotide metabolism. The effect of such mutations will be examined on the metabolism of pyridine nucleotides.